This invention provides expression vectors comprising multiple shear stress responsive elements and gene therapy methods for treating disorders related to or associated with vasculogenesis and/or angiogenesis by introducing the vectors in the vasculature having hemodynamic forces such as fluid shear stress forces. Further, this invention provides methods to monitor shear stress activities, to screen and select for target genes and methods of identifying and describing genes which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states. Lastly, this invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease and for the identification, monitoring and therapeutic use of compounds as treatments of cardiovascular disease.
The construction of blood vessels consists of two processes: vasculogenesis, the establishment of vascular network during embryogenesis from multipotential mesanchymal progenitors, and angiogenesis, the sprouting of existing blood vessels which occurs both in the embryo and in the adult (1-5). Endothelial cells are the major players in both processes, they migrate and proliferate and than assemble into tubes with tight cell-cell connections. Peri-endothelial cells are recruited to support the endothelial tube, providing maintenance and modulatory functions to the vessel. These cells are perycytes in the capillaries, smooth muscle cells in larger vessels and cardiac mycocytes in the heart.
The establishment and remodeling of blood vessels is controlled by paracrine signals, many of which are protein ligands that bind to modulate the activity of transmembrane tyrosine kinase receptors (1,3,6). Among these molecules are Vascular Endothelial Growth factor (VEGF) and its receptors, Angiopeitin 1 and 2 and their receptor (Tie 1 and 2), Basic Fibroblast Growth Factor (bFGF), Platelet derived Growth factor (PDGF), and Transforming Growth Factor (TGF). Recently, some of these molecules have been disrupted in the embryo (null-mice), or mutated in the adult. The animal models pointed to a certain order in the formation of the blood vessel, and further stressed the role of the endothelium in all stages of vessel formation. The dominant role of VEGF and its receptors in preliminary stages of angiogenesis and vasculogenesis has been demonstrated in null animals which die in early stages of embryogenesis even in heterozygous animals (7-9). Null animals lacking the Flk1 receptor had no endothelial cells, while null animals lacking the Flt1 receptor failed to form tubes. When the Tie2 receptor or its ligands angiopoietin 1 and 2 were disrupted (10-14), endothelial cells organized into a tube shape, but failed to recruit the periendothelial cells. Interestingly, similar phenotypes were observed in animals null in PDGF-B, TGF- and tissue factor (15-18) suggesting that the binding of angiopoietin to its receptor may lead to the secretion of these molecules from the endothelium, pointing again for the role of these cells in the various steps of vessel formation.
The scheme evolving suggests that the binding of VEGF to its receptors, plays an important role in the primary steps of vessel formation, differentiation, migration and proliferation of endothelial cells and the formation of the primary tube, while the binding of Angiopoietin1 to its Tie2 receptor mediate the maturation of the vessel, recruitment of peri-endothelial supporting cells and maintenance of vessel integrity and quiescence. These same molecules play an additional role in loosening the interaction of the endothelium with its matrix and support cells, enabling the sprouting of new vessels (6). Recently it was suggested that the binding of Angiopoietin 2 to Tie2 plays a role in regression of already existing vessels (6,13).
In mature blood vessels, endothelial cells, by virtue of their unique anatomical position, are constantly exposed to the fluid mechanical forces generated by the flowing blood (See, William R. Milnor in Chapter 6 entitled xe2x80x9cThe Normal Hemodynamic Statexe2x80x9d of the book entitled xe2x80x9cHemodynamicsxe2x80x9d, published by Williams and Wilkins, Maryland (1989) and U.S. Pat. Nos. 5,199,298, 5,052,228 and 4,926,696 for measuring shear stress. These hemodynamic forces, which include hydrostatic pressure, cyclic strain and frictional wall shear stress, constitute a special category of physical stimuli that, in addition to better characterized biochemical stimuli, can elicit important biological responses in the cells that compose the blood vessel wall (23). The non-random distribution of early atherosclerotic lesions observed both in natural disease processes in human and in experimental animal model has long been cited as suggestive evidence for the role these forces play in the pathogenesis of cardiovascular diseases (24,25). Both in-vivo studies and in-vitro experiments, using well defined model flow systems, have demonstrated that wall shear stresses can modulate various aspects of endothelial structure and function, changes that are mediated via up or down regulation of endothelial gene expression at the transcription level (26,27).
Hemodynamic forces and more specifically, fluid shear stress, regulate the expression of PDGF and TGF( (26,27). The activity of extracellular degrading enzymes and the levels of their transcripts are all induced in endothelial cells exposed to flow (23,27). Recently, it was demonstrated that NO is a down stream signaling molecule in angiogenesis induced by VEGF, but not FGF (35). Endothelial NO synthase is highly regulated in cells exposed to shear stress.
One of the intriguing and unanswered questions is the role that hemodynamic forces play in the formation and maturation of blood vessels in both vasculogenesis and angiogenesis. The role of these forces in vessel formation has been so far only suggestive (4,5). The formation of coronary collaterals was suggested to be affected by hypoxia. Although VEGF and its receptors , as well as PDGF-B, are all induced in hypoxic endothelial cells and myocytes in-vitro (29-32), several in-vivo models demonstrated that collateral growth occurs outside the hypoxic area. In the canine model collateral growth occurs in the epicardium (which is not hypoxic) and proceeds at the time when even the endocardium is not hypoxic anymore (33). Another example from the peripheral circulation, is the ischemic foot (as a result of femoral artery occlusion) where collaterals develop in both ischemic and more distant non-ischemic regions (34). During maturation of the vessel changes in the composition of the extracellular matrix occur, and tight junctions are formed (19,20). Are these changes stimulated by the flow of blood in the newly formed vessel? Furthermore, angiogenesis often involves massive sprouting of the already existing vessels, which is accompanied by the regression of some of the newly formed tubes (21,22). Is this balance (formation versus regression) affected by changes in the rate and pattern of blood flow through these newly formed tubes? Do changes in the pattern and magnitude of hemodynamic forces in big vessels affect the formation of smaller vessels (vasa vasorum) in the adventitia (37)? Answers to these questions are essential for better understanding the multiple steps of vasculogenesis and angiogenesis.
This invention provides expression vectors comprising multiple shear stress responsive elements. Also this invention provides gene therapy methods for treating disorders related to or associated with vasculogenesis and/or angiogenesis by introducing the vectors described herein in the vasculature having hemodynamic forces such as fluid shear stress forces. Hemodynamic forces, which include hydrostatic pressure, cyclic strain, and frictional wall shear stress, play an important role in the formation and maturation of blood vessels by regulating endothelial genes through Shear Stress Responsive Elements (SSRE) in promoters of endothelial shear stress responsive genes.
This invention provides a recombinant vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE). The Shear Stress Response Elements (SSRE) are nucleic acid sequences from promoters of growth factors, thrombogenic factors or angiogenic genes.
This invention provides a pharmaceutical composition comprising the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier.
This invention provides a method of stimulating endothelial cell proliferation comprising introducing/transfecting the endothelial cells with an effective amount of the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier wherein the Shear Stress Response Elements transcriptionally regulate endothelial cell gene expression, thereby stimulating endothelial cell proliferation. As contemplated herein, endothelial cells are vascular endothelial cells or capillary endothelial cells.
This invention provides a method of modulating vascular permeability in a mammal, comprising administering to said mammal an effective amount of the pharmaceutical composition comprising the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier, wherein the pharmaceutical composition is administered to the mammal in the vasculature with the proviso that the vasculature has shear stress forces, so as to permit the Shear Stress Response Elements to be activated by the shear stress and transcriptionally regulate endothelial cell gene expression, thereby modulating vascular permeability in the mammal.
This invention provides a method of stimulating the formation, maturation or regression of blood vessels of a subject, comprising administering to said subject an effective amount of the pharmaceutical composition comprising the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier, wherein the pharmaceutical composition is administered to the mammal in the vasculature with the proviso that the vasculature has shear stress forces, so as to permit the Shear Stress Response Elements to be activated by the shear stress and transcriptionally regulate endothelial cell gene expression, thereby stimulating the formation, maturation or regression of blood vessels.
This invention provides a method of modulating genes or proteins involved in vascular diseases which comprises administering to a subject with the vascular disease an effective amount of the pharmaceutical composition comprising the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier, wherein the pharmaceutical composition is administered to the subject in the vasculature with the proviso that the vasculature has shear stress forces, so as to permit the Shear Stress Response Elements to be activated by the shear stress and transcriptionally regulate endothelial cell gene expression, thereby modulating genes or proteins involved in the vascular diseases.
This invention provides a method of treating a subject having a vasculogenic and/or angiogenic disorder comprising administering to the subject an amount of the pharmaceutical composition comprising the vector comprising a multiple number of nucleic acids of Shear Stress Response Elements (SSRE) and a suitable diluent or carrier, wherein the pharmaceutical composition is administered to the mammal in the vasculature with the proviso that the vasculature has shear stress forces, so as to permit the Shear Stress Response Elements to be activated by the shear stress and transcriptionally regulate endothelial cell gene expression, thereby treating the subject having the vasculogenesis and/or angiogenesis disorder.
The present invention relates to a method for revascularization of ischemic tissues, development of collateral vessels and improvement of function in peripheral and myocardial ischemic tissue and enhancing the level of perfusion of blood to a target tissue. Also, the present invention relates to a method for treating a target tissue suffering from or at risk of suffering from ischemic damage, and a method of inducing angiogenesis in a target tissue.
This invention provides a method for screening test compounds for the ability to regulate endothelial cell expression or angiogenesis and/or vasculogenesis comprising: (a) contacting a endothelial cells with the compound to be tested; (b) determining the amount or expression of the endothelial cells or the amount of angiogenesis and/or vasculogenesis produced as a result of the test compound; (c) stimulating endothelial cells by introduction of the vector provided herein; (d) determining the amount or expression of the endothelial cells or the amount of angiogenesis and/or vasculogenesis produced as a result of the vector; (e) comparing the amount of angiogenesis and/or vasculogenesis produced as a result step (b) to that of step (d), wherein an increased amount of expression of the endothelial cells or the amount of angiogenesis and/or vasculogenesis of the test compound means that the test compound regulates endothelial cell expression angiogenesis and/or vasculogenesis.
This invention provides methods to monitor shear stress activities, to screen and select for target genes and methods of identifying and describing genes which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states and a method for the identification and therapeutic use of compounds as treatments of cardiovascular disease. Lastly, this invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficacy of compounds in clinical trials.